The present invention generally relates to microscopy systems, and relates in particular to coherent anti-Stokes Raman scattering (CARS) vibrational imaging.
CARS vibrational imaging has been shown to be a powerful tool in biology due to its ability to visualize biological samples with high sensitivity without the necessity of labeling with fluorophores. Despite its sensitivity, CARS microscopy is hampered, however, by the presence of a non-resonant background signal that is intrinsically generated along with the vibrational specific signal. CARS arises from the third order nonlinear susceptibility χ(3), which is a sum of a vibrationally resonant part χres(3), and a non-resonant electronic contribution χnr(3). The background overwhelms weak resonant signals and contaminates the signal through coherent mixing, preventing straightforward reconstruction of the resonant signal of interest. The existence of a non-resonant background is a primary limitation that limits further sensitivity improvement of CARS microscopy. For certain biological and clinical applications of CARS microscopy, complete suppression of the background and full extraction of the resonant signal are desirable.
In addition to the non-resonant background, there are several other limitations constraining the current state-of-the-art CARS microscopes. First, the CARS signal scales quadratically with the concentration of the molecular entities under study. This hinders a quantitative assessment of weak signals. A linear dependence of the signal on the concentration would benefit a quantitative analysis of the acquired images and spectra. Second, current state-of-the-art CARS microscopes do not offer the possibility of amplifying weak signals. Weak CARS signals are difficult to detect, and a means of amplifying these weak signals would be highly beneficial. Lastly, the resonant contrast results from the Raman activity of molecules. Even though spontaneous Raman cross-sections and spectra of a vast amount of biochemical compounds are well-documented in literature, the CARS signal cannot be directly compared with the Raman literature values. This difficultly in correlating between CARS signals and spontaneous Raman values further limits the application of CARS microscopy as a routine imaging tool.
Suppression of the non-resonant background in CARS microscopy by detecting the CARS signal in a reverse or backward direction is disclosed in U.S. Pat. No. 6,809,814. Systems disclosed therein, however, do not suppress the contribution from sub-wavelength sized non-resonant features in focus with nonlinear susceptibilities different from that of water. Such systems also do not provide for the amplification of weak signals, or provide a methodology for quantitatively relating the CARS signal to Raman cross-section values.
Suppression of the non-resonant background in CARS microscopy by using differently polarized pump and Stokes fields is disclosed in U.S. Pat. No. 6,798,507. Suppression of the background by use of an analyzer (polarizer) not only results in rejection of the non-resonant contribution of the signal, but also may attenuate the resonant contribution. Application of the polarization sensitive microscope may be, therefore, limited to those studies in which the resonant signal is particularly strong. Also, isolation of Imχres(3) is not possible with the polarization CARS microscope, preventing a direct comparison with Raman cross-sections.
There is a need, therefore, for a system and method for providing improved sensitivity of CARS microscopy, and in particular to provide CARS detection with improved imaging of weak CARS signals. There is also a need to provide for comparison of CARS signals with spontaneous Raman values for a variety of bio-chemical compounds.